1. Field of the Invention
The present invention relates to a method of analyzing a very small quantity of a wide range of analytes, especially biopolymers such as proteins or nucleic acids at high speed and in high resolution, and is an apparatus for this purpose. More particularly, it is an apparatus that relates to a microchip electrophoretic method that employs a microchip. The microchip is prepared by forming a groove for feeding a liquid onto the surface of at least one of a pair of transparent plate members. The other plate member is provided with holes in positions corresponding to the groove. These plate members are adhered to each other while positioning the groove inward, thereby forming a separation passage by means of the groove. By filling up the separation passage with a migration liquid, injecting a sample into one end of it, and applying a migration voltage across the separation passage, the sample is electrophoresed in the separation passage.
2. Description of the Background Art
In the case of analyzing a very small quantity of protein or nucleic acid, an electrophoretic apparatus, such as a capillary electrophoretic apparatus, is generally employed. In the capillary electrophoretic apparatus, a migration buffer is filled in a glass capillary having an inner diameter of not more than 100 .mu.m. A sample is introduced into one end, followed by the application of a high voltage across the capillary for migrating the analyte in the capillary. The capillary, having a large surface area with respect to its volume, i.e., having high cooling efficiency, allows application of a high voltage, and can analyze a very small quantity of sample, such as DNA, at high speed in high resolution.
However, the capillary is fragile due to its small diameter even though it is usually protected by a polyimide coating. Therefore, the user must be extremely careful when exchanging capillaries. For this reason a capillary electrophoretic chip, called a microchip, has been proposed. This microchip is formed by connecting two substrates, as described in D. J. Harrison et al., Anal. Chim. Acta 283 (1993), pp. 361 to 366. FIGS. 1A to 1C show an example of such a microchip. This microchip consists of a pair of transparent substrates (such as glass plates) 51 and 52. Intersecting electrophoretic capillary grooves 54 and 55 are formed on a surface of substrate 52, while reservoirs 53 are provided on substrate 51 as holes in positions corresponding to the ends of grooves 54 and 55 respectively.
When employing this microchip, substrates 51 and 52 are overlapped, as shown in FIG. 1C, so that an electrophoretic buffer solution is injected into grooves 54 and 55 from any reservoir 53. Subsequently, a sample is injected into reservoir 53, corresponding to one end of shorter groove 54, while a pair of electrodes are inserted between reservoirs 53, corresponding to both ends of groove 54, for applying a high voltage to the sample injection at a prescribed time. In this way, the sample is dispersed into groove 54.
Then, another pair of electrodes are inserted between reservoirs 53, corresponding to both ends of longer groove 55, for applying a migration voltage. The sample, which is present on intersection 56 between grooves 54 and 55, is electrophoresed in groove 55. A detector such as an ultraviolet visible light photometer, a fluorescent photometer, or an electrochemical detector, is located in a correct position to groove 55, for detecting separated components.
Conventional microchip electrophoretic apparatuses employing optical detectors for the detection of constituents use laser induced fluorescence detection as a detection means. No apparatus performs detection by absorption in an ultraviolet visible region. If ultraviolet visible absorption detection is to be applied, it is conceivable as a method of introducing incident light perpendicularly to a passage and detecting target components passing through a certain specific position of a detection part through means such as a photocell, thereby confirming separation in a similar way to a conventional capillary electrophoretic apparatus.
When light is introduced into a microchip perpendicularly to its surface as in case of capillary electrophoresis, an optical path length of only about 20 .mu.m can be obtained and this is about 2/5 of that in conventional capillary electrophoresis. Also in the conventional capillary electrophoresis, the optical path length (about 50 .mu.m) is so short that its inferior sensitivity is a serious drawback despite its high resolution. Although it is conceivable to swell a passage of a cell part in order to increase the optical path length, as observed in a bubble cell or the like, it is still difficult to implement a sufficient optical path length.